We are interested in understanding the role of the coiled-coil domain which is the specific region responsible for filament formation in nonmuscle myosin II (NMHC II, both A and B isoforms). Serial deletions were made from the carboxyl terminus of NMHC II and green fluorescent protein (GFP) was fused to the N-terminus of the truncated myosin. The GFP fusion proteins were then transiently expressed in HeLa Tet-Off cells (expressing only NMHC II-A), COS-7 cells (expressing only NMHC II-B) and HeLa-ATCC (expressing both NMHC II-A and II-B) to visualize filament formation of GFP-tagged truncated myosin. Both full-length myosin II-A and II-B formed filaments in all three cell lines. Deletion of the nonhelical region at the very end of myosin II-A (amino acids 1928-1961) and II-B (amino acids 1933-1976) did not have an effect on myosin filament formation. The myosin II-B homologue of the previously identified assembly competence domain (ACD) in skeletal myosin (Sohn et al., J. Mol. Biol., 266: 317-330, 1997) was required for filament formation. However, deletion of 10 amino acids upstream from the nonhelical region (amino acids 1918-1928) in myosin II-A significantly affected filament formation, suggesting that the myosin II-A homologue of the previously identified ACD was not enough to maintain filament formation in myosin II-A. An inducible cell line expressing a truncated myosin II-A, DeltaC170 (lacking amino acids 1791-1961), was also established in HeLa Tet-On cells. DeltaC170 did not form filaments and cells expressing DeltaC170 rounded up and detached after 3 days' culture. Furthermore, expression of DeltaC170 in COS-7 and HeLa-ATCC cells disrupted the stress fiber formation, suggesting that DeltaC170 can interfere with the function of endogenous myosin II, leading to cell death. We also ectopically expressed the transcription factor Pitx2a, one of the Pitx2 isoforms, in Hela cells using a tetracycline-inducible expression system and examined whether Pitx2a was capable of modulating the Rho GTPase signaling. Indeed, ectopic expression of Pitx2a induced actin-myosin cytoskeleton reorganization, leading to increased cell spreading, suppression of cell migration and strengthening of cell-cell adhesion. Beta-catenin and N-cadherin accumulated and localized to the sites of cell-cell contacts. Rho GTPases Rac1 and RhoA were activated and the dominant negative Rac1 mutant (N17Rac1), but not the dominant negative RhoA mutant (N19RhoA), inhibited the cell spreading and disrupted the localization of beta-catenin and N-cadherin to the sites of cell-cell contacts. Actin-myosin reorganization and cell spreading require PI 3-kinase activity. Pitx2a transiently induced the expression of Trio, a guanine nucleotide exchange factor for Rac1 and RhoA, which preceded cell spreading induced by Pitx2a. The expression of Trio was downregulated when the changes in cell spreading and cell morphology were observed. In addition, Pitx2a also induced cell cycle arrest at G0/G1, most likely due to the accumulation of the tumor suppressor proteins p53 and p21. The overall result of Pitx2a expression is a HeLa cell with less malignant properties.